Quantitative analysis methods on active electronic microarrays

ABSTRACT

The present invention presents techniques useful in methods for gene expression monitoring, and other nucleic acid hybridization assays, that utilize microelectronic arrays to drive the transport and hybridization of nucleic acids. Particularly, methods for normalizing the signals of individual microlocations by the use of an internal control sequence probe are provided. These methods are particularly useful for hybridization assays in which a quantitative comparison of the hybridization of several different sequences at a plurality of microlocations is desired, such as in gene expression analyses.

This application is a divisional of U.S. patent application Ser. No.09/710,200, filed on Nov. 9, 2000, now U.S. Pat. No. 6,379,897 Apr. 30,2002.

FIELD OF THE INVENTION

The present invention presents methods for gene expression monitoringthat utilize microelectronic arrays to drive the transport andhybridization of nucleic acids. Procedures are described for generatingmRNA expression samples for use in these methods from populations ofcells, tissues, or other biological source materials, that may differ intheir physiological and/or pathological state. Provided in the inventionare methods for generating a reusable nucleic acid transcript libraryfrom mRNA in a sample of biological material. In order to improve geneexpression monitoring on the microelectronic arrays, the transcripts areamplified to produce sample nucleic acid amplicons of a defined length.Because multiple sample amplicons may be selectively hybridized tocontrolled sites in the electronic array, the gene expression profilesof the polynucleotide populations from different sources can be directlycompared in an array format using electronic hybridizationmethodologies. Also provided in the invention are methods for detectingthe level of sample amplicons using electronically assisted primerextension detection, and utilizing individual test site hybridizationcontrols. The hybridization data collected utilizing the improvedmethods of the present invention will allow the correlation of changesin mRNA level with the corresponding expression of the encoded proteinin the biological source material, and thus aid in studying the role ofgene expression in disease.

BACKGROUND OF THE INVENTION

The human genome contains approximately 100,000 genes. These genes areexpressed at vastly different levels; the majority of species, over 90%,are present at low abundance, i.e. at five to fifteen copies per cell,while a few high abundance genes are expressed at thousands of copiesper cell. In addition to the different levels of basal expression, geneexpression is modulated in response to cell state, cell type,extracellular environment, disease, etc. Thus, information on changes inthe levels of genes will enable a greater understanding of thepathological and/or physiological state of the organism under conditionsof interest.

A number of methods currently exist for analyzing the expression levelsof different messenger RNA (mRNA) species. Subtractive hybridization wasused early in the history of monitoring of gene expression to analyzedifferences in levels of gene expression in different cell populations(Scott, et al.). This technique is not sufficiently sensitive to detectmessages present at low levels in a polynucleotide population.Representational difference analysis is a more recent modification thatincludes amplification after subtraction, in order to detect mRNAs thatare expressed at low levels (Hubank and Schatz). While this methodallows identification of differentially expressed messages that arepresent at low levels, the amplification step makes quantificationdifficult.

Adaptations of the polymerase chain reaction (PCR) have proven valuablein the field of gene expression. Reverse transcription coupled withcompetitive PCR (Competitive RT-PCR) involves co-amplifying a knownamount of an exogenous RNA competitor with the target mRNA sequence(Gilliland, et al.). The amount of target is extrapolated from atitration curve based on the concentration of competitor. Thedifficulties with this technique lie in the limited dynamic range of theassay and the tedium of constructing separate competitors for eachtarget of interest.

Real-time PCR is a powerful approach for gene expression monitoring. Theoriginal method detected accumulation of double stranded species duringamplification using ethidium bromide and an adapted thermocycler(Higuchi, et al.); detection of non-specific products was a drawbackthat was subsequently overcome by designing of probes that generatesignal only if the target of interest is amplified (Holland, et al.;Lee, et al.). This approach requires that the linear ranges ofamplification are similar for abundant internal controls and endogenoustarget mRNAs that may be present at much lower levels. In addition,primer design is critical and requires special software programs foroptimal efficiency.

Differential display PCR (dd-PCR) is also a PCR-based method that hasbeen adapted for monitoring gene expression. The original protocol usedsets of random, anchored primers to amplify all mRNAs in two differentcell populations; differences in levels are visualized by separating thePCR product on denaturing polyacrylamide gels (Liang and Pardee). Manyvariations on this original technique have been devised. In general,however, the PCR-based amplification of these methods results in a lackof quantitative correlation of band intensity with message abundance,variable reproducibility, and a high level of false positives. Resultsgenerated by dd-PCR must therefore be confirmed by other methods.

Serial analysis of gene expression (SAGE) is another technique for geneexpression monitoring. Short sequence tags that uniquely identify themRNA transcripts in a given cell population are isolated, concatenated,cloned and sequenced (Velculescu, et al.). The frequency of any giventag reflects the abundance of the corresponding transcript. Thistechnique, while powerful, is rather complicated, requires generationand analysis of large amounts of sequence data, and the amplificationevent can skew quantitation.

The most recent developments in the field are in the area of microarrays(Schena, et al.; DeRisi, et al.; Zhao, et al.). Gene-specific probes areindividually arrayed on a solid matrix and incubated with labeled cDNAsfrom control and experimental populations. Comparison of the intensityof probe hybridization with cDNA targets from the distinct samplesreveals differences in expression of the corresponding mRNAs. Becausethese arrays are hybridized passively in a low stringency buffer,differences in availability of relevant target sequences to thecomplimentary probes on the array may not be uniform. In addition,hybridization characteristics of each probe will vary, due to T_(m)considerations and the affinity of probe-target interactions. Therefore,while these high-density microarrays offer high-throughput, thehybridization kinetics may not be optimal for all different probe-targetcombinations.

Although great strides have been made in methods to detect alterationsin gene expression, each of the procedures has drawbacks as well asadvantages, as indicated above. All of the above approaches are eithertime consuming, complicated, labor intensive, or a combination of allthree. Rapid, sensitive approaches that allow simultaneous monitoring ofmultiple mRNAs are still needed.

SUMMARY OF THE INVENTION

The present invention provides a method that allows efficient electronichybridization of amplified nucleic acids generated from target mRNAs tocomplementary probes in a microarray format. The use of electric fieldsto transport and drive hybridization of nucleic acids allows the rapidanalysis of polynucleotide populations. Utilizing electronichybridization devices, such as those described in U.S. Pat. No.5,605,662, hybridization assays may be accomplished in as little as 1-5minutes. Additionally, because each site on the microarray isindividually controlled, targets from different samples can be analyzedon the same matrix under optimized conditions, an aspect unique to thistechnology. By improving the use of electronic hybridization methods anddevices in gene expression monitoring applications, the disclosedmethods will dramatically increase the ability of those in the art torapidly generate gene expression information with a minimum ofsequence-specific optimization.

The methods of the invention facilitate the use of electronicallyhybridized gene expression monitoring for both research and clinicalapplications in several ways. First, through the use of shortenedamplicons of uniform size, the methods of the invention allow the rapid,simultaneous monitoring of dozens of genes in comparative andquantitative procedures with minimal interference fromcross-hybridization and secondary structure formation. Because theindividual test sites in the electronic array may be selectivelycontrolled, several samples may be screened on the same microarray inthe same experiment. Preferred embodiments of the method for determiningthe level of mRNA expression in the cells of a biological sample includethe steps of (a) isolating mRNA from at least one biological sample, (b)quantitatively amplifying from the isolated mRNA population at least twogene sequences of interest to produce shortened amplicons of less thanabout 300 bases in length, (c) electronically hybridizing the ampliconsto at least two probes bound to a support at predetermined locations,and (d) determining the amount of each amplicon hybridized to each probeat the predetermined locations.

Although several equally desirable embodiments of the general method ofthe invention are provided, it is preferred that the quantitativeamplification step of the method comprise a linear amplification step inwhich the sequences of interest are amplified from a fixed amount oftemplate generated from the reverse transcription of the mRNA populationisolated from the biological sample. Exemplary preferred processesinclude single primer DNA polymerase amplification and in vitrotranscription amplification. The amplicons are preferably shortenedduring the amplification process through the use of matched sets of“bookending” primers which generate amplicons of a defined length, or bythe utilization of an endogenous or introduced type IIs endonucleasesite to cleave the amplicons at some point in the amplification process.The shortened amplicons produced for use in the methods of the presentinvention are preferably about 50 to about 300 bases in length, morepreferably about 50 to about 200 bases in length, and most preferablyabout 50 to about 100 bases in length.

As the electronic hybridization processes of the method may be carriedout on arrays of individually electronically controlled test sites,multiple genes may be monitored in multiple samples during a singleexperiment on the same electronic array device. At least two, at leastten, and even fifty or more samples may be assayed in a singleexperiment. Similarly, at least, 5, 10, 20, 40, or 50 or more differentgenes may be simultaneously monitored in an experiment. As electronicmicroarray devices with tens of thousands of test sites have beenproduced, and the electronic hybridization process can be completed inas little as 1-5 minutes, an experiment in which 80 genes are monitoredin 100 different samples sequentially hybridized to rows of test siteson the array may be completed in a few hours.

Detection methods which may be used in the gene expression monitoringmethods of the present invention include all commonly employed nucleicacid hybridization interaction detection methods such as primerextension labeling, amplicon labeling, reporter probe detection, andeven intercalating dyes. The detectable moiety in these labeling methodsmay be a fluorophore, chemiluminescent, colorigenic, or other detectablemoiety. Fluorophore moiety labels are preferred for use in the presentinvention because of their widespread availability and relative ease ofuse.

In as second aspect, the present invention provides methods for the useof reusable bead libraries produced from mRNA samples to extend theeffective amount and life of precious biological and patient samples byallowing re-amplification of the same sample nucleic acids. Preferredembodiments of this method of the invention include the steps of: (a)isolating mRNA from a patient sample; (b) reverse transcribing a cDNAlibrary from the mRNA isolate; (c) amplifying the cDNA library with aprimer containing an upstream RNA polymerase promoter site upstream of asequence specific for the mRNA of interest and a fill-in primer, whereinat least one of the primers comprises an affinity moiety; (d) bindingthe amplification products from (c) to a solid support coated with anaffinity-binding moiety; (e) utilizing the bound amplification productsas a template for an in vitro transcription reaction; (f) separating thein vitro transcription products from step (e) from the amplificationproducts bound to the solid support; and (g) utilizing the boundamplification products from step (f) as a template for at least oneadditional in vitro transcription reaction, wherein the amount of invitro transcription product produced is not significantly less than thatproduced in step (e).

In more preferred embodiments, steps (f) and (g) are repeated one, two,or even three or more times. As observed by applicants, the amount oftranscript produced in successive rounds of in vitro transcription doesnot decrease significantly as compared to the amount of transcriptproduced in the proceeding round. Preferably, at least about 70%, morepreferably at least about 80%, and most preferably at least about 90% ofthe amount of transcript produced in a preceding round of transcriptionis produced in a succeeding round.

Preferred affinity moieties for use in the reusable library method ofthe invention include biotin, haptens, and antigenic moieties. Biotin isparticularly preferred, and in embodiments where biotin is the affinitymoiety, streptavidin and avidin are preferred affinity-binding moieties.Preferred solid supports for use in the reusable library method includebeads, microtiter wells, pins, and the like. Exemplary preferred beadsinclude paramagnetic beads, polymer beads, and metallic beads.

In a third aspect, the present invention provides rapid detectionmethods for detecting the hybridization of target sequences to theelectronic microarray without the need for additional reporter probes,or labeling of the target sequences, using primer extension reactions.Preferred embodiments of this method of the invention comprise the stepsof (a) electronically hybridizing a nucleic acid in a sample to anucleic acid probe bound to a support at a predetermined location; (b)utilizing the hybridized nucleic acid as a template in a nucleic acidpolymerase reaction to extend the bound probe, thus incorporating alabeled nucleotide into the extended probe; and (c) detecting thelabeled nucleotide incorporated into the extended bound probe. Preferredlabeling moieties for the labeled nucleotide include fluorescentmoieties, colorigenic moieties, chemiluminescent moieties, and affinitymoieties. Fluorescent moieties are particularly preferred. Nucleic acidpolymerase reactions which may be used in the method include DNApolymerase reactions (where the hybridized nucleic acid is DNA) andreverse-transcriptase reactions (where the hybridized nucleic acid isRNA).

A fourth aspect of the present invention is a method of providing aninternal control for individual test sites on an electronicallycontrolled microarray for use in nucleic acid hybridization reactionassays for determining the presence of nucleic acid sequences innucleic-acid-containing samples. Such internal controls are useful forreal-world applications of microarray technology because of the inherentirregularities introduced by the microfluidics systems which distributethe sample and reagents to the surface of the microarray. Preferredembodiments of the method comprise the steps: (a) attaching a mixednucleic acid probe consisting of a first nucleic acid probe specific fora first nucleic acid sequence known to be present in the sample (e.g.,endogenous or spiked), and a second nucleic acid probe specific for asecond nucleic acid sequence of interest to a first test site on theelectronically controlled microarray; (b) attaching a mixed nucleic acidprobe consisting of the first nucleic acid probe and a third nucleicacid probe specific for a third nucleic acid sequence of interest, whichmay be the same as or different than the second nucleic acid sequence,to a second test site; (c) electronically hybridizing the sample nucleicacids to the nucleic acid probes on the first and second test sites; (d)specifically detecting the extent of hybridization of the sample nucleicacids to the first nucleic acid probe at the first and second testsites; (e) specifically detecting the extent of hybridization of thesample nucleic acids to the second and third nucleic acid probes at thefirst and second test sites; (f) comparing the hybridization valuesobtained for the first nucleic acid probe at the first and second testsites to obtain a normalization factor; and (g) normalizing thehybridization values obtained in (e) for the second and third probesusing the normalization factor obtained in (f).

Preferred embodiments of the internal control methods of the inventionutilize an endogenous “housekeeping” gene sequence, which is known to bemaintained at a steady-state level across the relevant sample celltypes, as the first control sequence. Alternatively, exogenous nucleicacid sequence may be added to the sample at known concentrations. Thedetection methods utilized to specifically detect the hybridization ofthe sample nucleic acids to the first and the second and third nucleicacid probes may be independently chosen from any standard detectionmethod, including the labeling of amplified sample nucleic acids throughsequence specific primers, primer extension detection, hybridization ofreporter probes to bound sample nucleic acids, or a combination of thesemethods. In order for hybridization to the first nucleic acid probe tobe distinguishably detectable from hybridization to the second and thirdnucleic acid probes, it is desirable to use two easily distinguishabledetectable moieties. Preferred detectable moieties for use in theinternal control method are fluorescent moieties with different emissionwavelengths. Alternatively, the extent of hybridization to the first(control) probe may be determined first using a detectable moiety afterperforming a first selective labeling method, and then the extent ofhybridization to the second and third probes determined after a secondselective labeling method with the same detectable moiety by determiningthe increase in the detectable signal.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: A schematic of two alternative methods for generating shortenedamplicons of a defined size for use in the mRNA gene expressionmonitoring methods of the invention.

FIG. 2: A schematic of an electronically addressable microarray devicewhich is similar to the device used in the Examples. The depicted deviceis described in detail in U.S. Pat. No. 5,605,662. Note that in thisdevice the test sites on the array are defined in the plane of the arrayby the electrodes, but are separated from the electrodes by a permeationlayer, to which nucleic acid probes may be attached.

FIG. 3: Shown are the agarose gel electrophoresis-separated RNA productsof a 1^(st), 2^(nd), and 3^(rd) round of in vitro transcription from thesame double stranded amplified cDNA library immobilized on magneticstreptavidin-coated beads. The far left lane contains a molecular sizemarker.

FIG. 4: Shown are grayscale hybridization fluorescence data for fiveseparate gene probes, using 50, 100, 250, and 500 nucleotide amplicons.As can be seen from the best results were obtained using amplicons ofaround 100 bases in length.

FIG. 5a: Shown are grayscale hybridization fluorescence data for serialdilutions of amplified sample plasmid nucleic acids, hybridized to aβ-la capture probe. NS denotes no capture sequence attached to the testsites in the far right column. Note the reproducibility of thehybridization signal across the rows of the electronic array.

FIGS. 5b & 5 c: Shown are grayscale hybridization fluorescence data fordefined amounts of sample nucleic acid sequence, and a graphicalrepresentation of a concentration curve derived from these data.

FIG. 6: Shown are grayscale hybridization fluorescence data for fourseparate gene probes and a control sequence (NS) on twelve test sites ona NanoChip™. mRNA was isolated from LPS induced and uninduced monocytes;amplification, hybridization and reporting were carried out as describedin Example 5. The treated monocytes clearly show an altered mRNAexpression level in IL1, TGFβ, and JUN, while the expression level ofIL6 is only slightly increased.

FIG. 7: A graphical illustration of the results shown in FIG. 6.

DEFINITIONS

As used herein, “nucleic acid” refers to any nucleic acid polymer,including deoxyribonucleic acid and ribonucleic acid polymers, includingmodified DNA or RNA, including synthetic oligonucleotides, peptidenucleic acids (PNA), pyranosyl nucleic acids (pRNA), and derivatives ofthese nucleic acids.

“Amplicon,” as used herein, denotes an amplified nucleic acid sequencewhich may be either RNA or DNA, depending on the amplification methodused (e.g., DNA PCR produces a DNA amplicon, while in vitrotranscription produces an RNA amplicon.) The amplified nucleic sequencecomprises either the same or complementary sequence as that of theoriginal nucleic acid sequence which was amplified. “Shortened amplicon”denotes an amplicon which contains a portion of the full endogenousnucleic acid sequence rather than the whole sequence.

“Electronic hybridization” means the use of electric fields toconcentrate nucleic acids within a certain area in order to reduce thetime necessary to hybridize complementary nucleic acids. Electronichybridization is advantageously performed using electronicallycontrolled microarray devices, such as those described in U.S. Pat. No.5,605,662. These devices comprise individually controllable electrodeswhich are covered by a permeation layer, or layer of material whichpermits ion exchange between the buffer and the electrode, but whichinhibits contact between nucleic acids in the buffer and the electrode.Nucleic acid capture probes may be advantageously attached to bindingmoieties within or on the permeation layer of these electronic arraydevices.

“Gene,” as used herein, means an organismal nucleic acid sequenceencoding a protein, such as genomic DNA or mRNA (including splicingvariants), or a copy or portion thereof produced by molecular geneticmanipulations, such as cDNA, in vitro transcribed RNA, cloned DNA, etc.As used herein “target” generally refers to a gene of interest.

“Amplification,” as used broadly herein, encompasses various processesand sets of processes (including: reverse transcription, DNA polymerasereactions, ligase reactions, in vitro transcription, and combinationsthereof) for copying nucleic acids (either in their originaldeoxyribonucleotides or ribonucleotides, or other natural or artificialnucleotides) through molecular genetic manipulations. “Linearamplification” or “quantitative amplification” refers to processes andsets of processes for producing copies of nucleic acids from asubstantially constant amount of template, so that the number of copiesof the nucleic acid, or its complement, increases linearly as a functionof time, rather than exponentially.

DETAILED DESCRIPTION OF INVENTION

The methods described are designed to determine the abundance of targetgenes in a distinct polynucleotide population, particularly as comparedto the abundance of those target genes within a different polynucleotidepopulation. The methods of this invention are particularly suited forgathering data to correlate differences in expression patterns withspecific physiological and/or pathological states. Electronic nucleicacid hybridization techniques and devices (such as those described inU.S. Pat. No. 5,605,662, incorporated herein in its entirety), arecapable of dramatically reducing the time necessary to perform variousnucleic acid hybridization procedures, and are thus an extremely usefultool for a wide variety of biological assay methods. The methods of thisinvention enable those of skill in the biochemical arts to more fullyexploit this tool to study the expression of genes in organisms undervarious physiological conditions.

In the methods of this invention, target genes are quantitativelyamplified from mRNA populations derived from any biomaterial, including,but not limited to, cells from unicellular organisms, cells derived fromin vitro cultured cell lines, and cells or tissues from multicellularorganisms. In one preferred embodiment, the amplified target genes(amplicons) are enzymatically shortened using a type IIs restrictionendonuclease. In another preferred embodiment, generation of shortenedamplicons is mediated via sequence specific oligonucleotides which“bookend” the amplified sequence. Shortened amplicons are analyzed byelectronic hybridization to capture probes comprising complementarynucleic acids that are specific for each target gene of interest. Thesecapture probes are preferably immobilized in a microarray format.Detection of the hybridized targets may be performed by primer extensionof the capture probe, or by hybridization of a second complementaryspecies that is labeled, or by other means.

An important aspect of the invention is the generation of target nucleicacid sequences which are of similar size and/or reduced length. Theefficiency and uniformity of electronic hybridization is increased whentargets are limited in size. In addition, efficient analysis ofdifferent targets is facilitated if the target species are similar insize because variations in electronic transport and hybridization ofdifferent targets are reduced. Further, when the target(s) beingexamined comprise single stranded nucleic acids, particularlyribonucleic acids, secondary structure considerations are minimized byreduced length. Gene expression monitoring on electronic microarrays isthus improved by utilization of shortened targets of relatively uniformsize, as is demonstrated in the data of Table 1.

mRNA Isolation and cDNA Library Preparation

The initial step in the subject invention is isolation of a sample ofpolynucleotides, usually mRNA, from populations of interest. Thepolynucleotide populations may be derived from a variety of sources,including but not limited to different cell types from in vitro culturedcell lines or different cell types from organs or tissues ofmulticellular organisms, or the same cell type from different organismsof the same species. The polynucleotide populations may also be derivedfrom the same cell type from in vitro cultured cell lines or from anorgan or tissue of a multicellular organism, at different stages ofdevelopment, disease, or treatment.

After isolation by standard means well known to those of skill in themolecular genetics arts, reverse transcription is performed to generatesingle-stranded cDNA from the mRNA population. cDNA synthesis can beperformed by any method known to those of skill in the art. The variousdNTPs, buffer medium, and enzyme with reverse transcriptase activity maybe purchased commercially from various sources. Applicants have foundthe Superscript™ enzyme system to be well suited for the production ofan initial cDNA library. First strand synthesis may be directed by anoligo(dT) primer that hybridizes to all polyadenylated RNA species. Theoligo(dT) primer is usually 10-30 bases long, more preferably 12-18bases long, and may comprise a mixture of primers of different lengths.Other suitable polythymine primers include the non-replicable dT primerdescribed in U.S. Pat. No. 6,027,923, and oligo(dT)_(n)V (V=A, C, or G)primers.

Quantitative Shortened Amplicon Generation: Primer Extension and invitro Translation Techniques

In transcription-amplification embodiments of the invention, thegeneration of amplicons is accomplished through the use of a chimericoligonucleotide specific to each target of interest. Following cDNAsynthesis, the target(s) of interest may be linearly amplified by primerextension of a chimeric oligonucleotide(s) using DNA polymerase. Linearamplification either by primer extension, as here, or by other means(such as in vitro transcription, used below), is necessary in order toallow quantitative comparisons between different samples. The sequencespecific portion of the chimeric oligonucleotides are sufficientlyspecific as to anneal to the complementary target sequence underconditions of primer extension by a polymerase reaction (DNA polymeraseif produced from a cDNA library), which are familiar to those of skillin the molecular biology arts. In order to generate target amplicons forthe gene sequences of interest, one chimeric oligonucleotide specific toeach target of interest is used. These oligonucleotides contain an RNApolymerase promoter sequence at the 5′ end; this sequence may be theconsensus binding site for T7, T3, SP6, or another RNA polymerase.Adjacent to the RNA polymerase promoter site is a sequence specific tothe target gene of interest. This gene-specific sequence is notrestricted in length; however, to promote efficient annealing of thechimeric oligonucleotide(s) to the target(s) of interest, thegene-specific sequence will preferably be between 15 and 35 bases inlength. The oligonucleotides may contain a biotin moiety at the 5′ end,or may be unmodified. In embodiments utilizing a type IIs endonucleasefor shortened amplicon generation, the chimeric primer will alsocontain, at the 3′ end, the recognition sequence for a type IIsrestriction endonuclease.

The sets of chimeric oligonucleotides that are used in targetpreparation will generally represent at least two distinct targetspecies but may represent 10, 20, 40, or even up to 50 distinct targetspecies. Above about 50 different target species, amplificationefficiency and the quantitative nature of the results may becompromised, utilizing currently available amplification techniques.However, the use of the present invention with improved amplificationtechniques to measure, potentially, 80, 90 or over 100 specific genessimultaneously is also contemplated by the present methods. The chimericoligonucleotides are typically chosen to represent targets that areknown or suspected to be differentially expressed under the experimentalconditions (e.g., cell type, or two different stimuli for a single celltype), or may represent targets for which no expression data areavailable. One of the chimeric oligonucleotides in the set is chosen toallow analysis of levels of a housekeeping gene that is known to beexpressed at similar levels in the different conditions being examined,or of an added exogenous control sequence.

Examples of commonly used housekeeping genes include glyceraldehydephosphate dehydrogenase (GAPDH), β-actin, and ribosomal RNAs. Controlscan also comprise exogenous sequences that are added into the startingmaterial at known concentration and processed along with the targets ofinterest. An example of such sequence is the β-lactamase gene, aprokaryotic gene that confers ampicillin resistance. (For review, seeReischl, U. and Kochanowski, B. (1999) “Quantitative PCR” QuantitativePCR Protocols (pp.3-30). Humana Press., Totowa, N.J.; and also Ferre F.(1992) Quantitative or semi-quantitative PCR: Reality versus myth. PCRMethods Appl. 2, 1-9.) The selection of a particular control sequencefor use in an application of the described methods will be casespecific, depending on the organism used, the particular cells studied,and the familiarity of the researcher with a particular constitutivelyexpressed sequence. However, using the guidelines presented, one ofordinary skill in the art could readily select an endogenous orexogenous sequence for use as a control in the present methods.

Single-primer extension may be directed by a thermophilic DNApolymerase, usually a 3′-5′ exonuclease-minus derivative polymerase,e.g. Vent®_(R)(exo−) DNA polymerase. Multiple copies of the target(s)are generated during the extension reaction, in which repeated cycles ofdenaturation, oligonucleotide primer annealing, and DNApolymerase-directed primer extension are performed. Following generationof multiple single-stranded copies of target(s) from the cDNA pool, thecomplementary strands are generated. In one embodiment, generation ofthe complementary strand is mediated by a common primer that binds toall amplified targets. This primer may be the oligo(dT) oligonucleotideused for first strand cDNA synthesis, a fraction of which is carriedover from the first strand cDNA synthesis reaction into the primerextension reaction. Alternatively, the primer may be a mixture of randomshort polynucleotide sequences, e.g. random hexamer primers. In thisembodiment, the primer is allowed to anneal at a temperaturecorresponding to the Tm of the primer used for second strand synthesis.In another embodiment, a gene-specific oligonucleotide for each targetof interest is utilized, yielding a “bookended” product, as describedfurther below. This primer will generally be 15-30 bases in length andwill hybridize at a specific site within the amplified target such thatthe second strand produces an amplicon of a defined length. The primersmay contain a biotin moiety at the 5′ end, or may be unmodified. Primersare allowed to anneal at or slightly below the lowest Tm among theprimers in the set. After allowing sufficient time for the primers toanneal, the temperature is increased to allow efficient extension of theprimers by the thermophilic DNA polymerase.

Shortening of Amplicons by Type IIs Endonuclease Cleavage or Bookending

Amplicons for use in the present invention preferably are less thanabout 300 bases, more preferably less than about 200 bases, and mostpreferably less than about 100 bases. Because the amplicon must be of asufficient size to hybridize specifically to the capture probe,amplicons are preferably greater than 15 bases in length, and are morepreferably at least about 50 bases in length. In addition, although theamplicons for use in the methods of the invention may differsignificantly in length, it is preferred that they be of similar length,preferably differing in length by less than 20 bases, and morepreferably differing in length by less than 10 bases. The generation ofshortened amplicons may be accomplished through the use of Type IIsendonucleases, or by the use of primers designed to “bookend” a stretchof the target sequence. Although both methods are described below, othermethods may be devised by those of skill in the art. The use ofshortened amplicons made by alternative amplification strategies in themethods of the present invention is contemplated to be within the scopeof those methods, so long as a shortened, quantitatively amplifiednucleic acid is produced from the sample mRNA.

In a first strategy, shortening of the target nucleic acid sequences isperformed by digestion with a type IIs restriction endonuclease. TypeIIs restriction endonucleases cleave at a defined distance from therecognition site, thus producing a gene-specific “tag” that cansubsequently be used to isolate and quantify each target. For thisembodiment, a type IIs restriction endonuclease recognition site isincorporated at or near the 3′ end of the chimeric oligonucleotide usedfor amplification, supra. Where possible, this sequence will be presentin the target(s) of interest, and the chimeric oligonucleotide(s) willbe designed to utilize the endogenous restriction endonucleaserecognition sequence. In this case, the gene-specific portion of theoligonucleotide(s) will anneal to the restriction endonucleaserecognition site and sufficient adjacent sequence 5′ of the recognitionsite to produce an amplicon of the desired length upon cleaving.

Where an endogenous type IIs restriction enzyme recognition site is notpresent in the target of interest, a sequence within the target will bechosen that resembles the desired site as closely as possible, withpreference given to sequences that perfectly match the 3′ end of theendonuclease recognition site. The chimeric oligonucleotide will then bedesigned to generate the desired restriction endonuclease recognitionsite by altering the target sequence during primer extension. Suchalterations may include changing, inserting or deleting nucleotides asnecessary to generate a type IIs restriction endonuclease recognitionsite into the amplified target.

The proximity of the restriction endonuclease recognition site to the 3′end of the chimeric oligonucleotide will in part be determined bywhether a site exists within the targets of interest or whether suchsite must be generated within the chimeric oligonucleotide. If anendogenous site is used, the chimeric oligonucleotide may end at thelast base pair of the restriction endonuclease recognition site or mayinclude additional target-specific sequence. In the case where thetarget sequence must be altered to generate a restriction endonucleaserecognition site, the chimeric oligonucleotide will generally, althoughnot necessarily, extend beyond the restriction endonuclease recognitionsite in order to permit the mutated oligonucleotide sequence toefficiently anneal to the target. The extension, if any, will preferablybe only a few bases in length, in order to ensure that sufficient targetsequence is retained upon cleavage by the Type IIs endonuclease.

Digestion with the type IIs restriction endonuclease will yield targetfragments that contain the RNA polymerase promoter sequence followed bythe target-specific sequence contained within the chimeric primer, plusthe target-specific sequence that lies between the type IIs restrictionenzyme recognition site and the type IIs restriction enzyme cleavagesite. If the chimeric oligonucleotide contains a biotin moiety at the 5′end, the digested fragments may at this point be isolated usingstreptavidin-coupled magnetic beads; alternatively, the Vent-amplifiedmaterial can be applied to the streptavidin-coupled magnetic beads forpurification prior to restriction enzyme digestion. If a biotinylated 3′gene-specific oligonucleotide is used to fill in the amplifiedsingle-stranded products, targets can be applied to streptavidin-coupledmagnetic beads prior to digestion and thereby isolated from anynonspecific materials. Subsequent to enzymatic digestion, the 5′ ends ofthe targets can be isolated away from the bead-bound 3′ ends of theamplified targets.

Subsequent to digestion, target fragments are used as templates in invitro transcription reactions mediated by a RNA polymerase, the promotersequence for which was incorporated via the chimeric oligonucleotide.Standard methods for in vitro transcription are known to those of skillin the art. In the case where biotinylated chimeric oligonucleotideswere used to generate amplified target, multiple rounds of in vitrotranscription can be performed; the bead-bound templates can also bestored for future in vitro transcription reactions. In vitro transcribedtemplates are then cleaned and desalted by any standard methodincluding, but not limited to, gel filtration columns.

In an alternative strategy, shortened targets are generated viaoligonucleotide primers rather than by endonuclease digestion. In thecase where oligo(dT), random primers, or gene specific oligonucleotideswere utilized to fill in the single-stranded amplified target(s), invitro transcription reactions are performed after the fill-in reaction.When a gene-specific oligonucleotide is used to generate the secondstrand, this primer is designed to hybridize to a sequence within thetarget at a defined length from the 5′ end, generally less than about300 base pairs away. In this “early bookending” embodiment, the RNAproduced in the in vitro transcription reaction is sufficientlyshortened such that the amplified material can be directly analyzed byelectronic hybridization. Alternatively, the shortened RNA products maybe subjected to another round of cDNA production and DNA polymerasemediated amplification utilizing gene-specific or random primers.

In the case where oligo(dT) or a set of primers of random sequence isutilized to fill in the amplified target(s), in vitro transcription willyield RNAs of variable length, some of which may be too short to berecognized by the capture oligonucleotide used in electronichybridization, as described infra, and some of which may be too long tobe efficiently electronically transported and hybridized. In order toimprove detection of these target(s), the RNA derived from the in vitrotranscription reaction is used as a template for a second cDNA synthesisreaction. For this method, a gene-specific oligonucleotide specific foreach target of interest is used to prime cDNA synthesis. This primerwill generally be 15-30 bases in length and will hybridize at a specificsite within the amplified target such that the second strand is of adefined length, generally less than about 300 base pairs, morepreferably about 50 to about 300 bases, more preferably from about 50 toabout 200 bases, and most preferably from about 50 to about 100 bases.This reverse transcription reaction is performed using reagents andmethods known to those of skill in the art, as described supra. Theproducts of this reaction will comprise shortened amplicons for eachtarget of interest

Electronic Hybridization of Shortened Amplicons

Analysis of target(s) is performed on microarrays using electronichybridization. The specific electronic hybridization procedures andprobe attachment will vary slightly from device to device, and may bemodified from the discussion below by one of ordinary skill in thebiochemical arts. For the purposes of illustration, hybridization anddetection procedures will be described with reference to the NanoChip™electronic hybridization device, similar to that pictured in FIG. 2.However, other electronically addressable devices for microscalebiochemical reactions may be utilized to carry out the methods of theinvention.

Capture probes are immobilized via interaction with a permeation layeron the electronic microarray surface, as described in U.S. Pat. No.5,605,662 , incorporated fully herein by reference. The stableinteraction of the probes may be accomplished by a variety of differentmethodologies including, but not limited to, streptavidin-biotininteractions in which the capture probes contain a biotin moiety at the5′ end and streptavidin is incorporated within the permeation layer.

Biotinylated capture probes specific to each target of interest areimmobilized at different position, or test site, on the microarray usingelectric field transport (i.e., electronically addressed to the testsites). When primer-extension detection methods will be used to quantifyhybridized amplicons, the biotin label should not be on the 3′ phosphateof the capture probe, so that the phosphate is available for extensionin the polymerase reaction. In embodiments where Type IIs endonucleaseshortening is used, these capture probes are designed to becomplementary to the target sequence that is between the type IIsrestriction endonuclease recognition site and the type IIs restrictionendonuclease cleavage site. The capture probes may contain additionaltarget-specific sequence including the type IIs restriction endonucleaserecognition sequence and upstream sequence. In embodiments wherebookending primers are used, capture probes are designed to hybridize toa region flanked by the binding sites for the chimeric oligonucleotideand the gene-specific oligonucleotide used to fill in single-strandedamplified products and/or generate cDNA. Generally the capture probeswill comprise about 18 to about 30 bases, although shorter or longercapture probes may be utilized with electronic hybridization procedures.Capture probes may include non-amplicon-complementary sequences for usein zip-code addressing of the probes, or for other purposes (e.g.,restriction endonuclease sites for selective cleavage).

In a preferred embodiment, a capture probe complementary to a controlsequence is addressed to one or more location(s) on the microarray. Thiscontrol sequence may be a housekeeping gene that is expressed at similarlevels under the different conditions being examined, or may be anexogenous sequence which is added to the sample nucleic acid mixtureprior to or after amplification. In a preferred embodiment, the captureprobe complementary to the housekeeping gene is combined with eachtarget-specific capture probe, and the two capture probes areco-addressed to a given position(s) on the microarray. This embodimentallows normalization within each position of the microarray, allowingbetter quantitative results. The microfluidics utilized to feed a samplenucleic acid solution introduce variations in exposure of eachindividual test site to the sample nucleic acid solution because of flowpatterns, pooling, etc. Thus, the use of an internal control probe ateach test site position allows for the normalization of assay data foreach individual test site microenvironment.

The pool of shortened amplicons (in vitro transcribed targettemplate(s), or cDNA copies thereof) is subsequently electronicallyhybridized to the immobilized capture probes. Because sites on the arrayare electronically controlled, hybridization can be restricted to asubset of locations within the microarray. Thus, different shortenedamplicon pools, derived from different samples, can be analyzed on thesame microarray. For these experiments, one pool of shortened ampliconsfrom a given condition, cell type, or other source is injected into theelectronic hybridization device, electronically transported to a subsetof locations within the microarray, and allowed to hybridize withspecific immobilized capture probes at those locations. Subsequently,this target pool is removed, unbound nucleic acids are washed away, anda second pool of targets from a different condition, cell type, or othersource is injected and electronically transported to a distinct subsetof locations within the microarray that contains the same immobilizedtarget-specific capture probes. This second target pool is allowed tohybridize and unbound material is removed by washing. Subsequently,hybridized target species are detected by one of a variety of reportingmethodologies.

This procedure, which allows direct analysis of a set of targets fromdifferent target samples on the same microarray, represents a particularstrength of electronic hybridization in the area of gene expressionprofiling. Using the methods of the invention, it is easy for one ofskill in the art to assay at least two, at least ten, and even fifty ormore samples in a single experiment. The results of this process areillustrated in FIG. 6, which shows the hybridization of two groups ofmRNA amplicons on the same chip: one sample was isolated from monocyteswhich have been treated with lipo-polysaccharide, and the other samplewas isolated from untreated monocytes. Alternatively, differentelectronic microarrays can be used to analyze expression patterns indifferent amplicon samples.

Detection of Hybridized Amplicons on Electronic Microarrays

Following electronic hybridization of target(s) to immobilized captureprobes, the bound target(s) may be detected by several means, includinginclude all commonly employed nucleic acid hybridization interactiondetection methods such as primer extension labeling, amplicon labelingpreferably through labeled sequence-specific primers), reporter probedetection, and even intercalating dyes. The detectable moiety in theselabeling methods may be a fluorophore, chemiluminescent, colorigenic, orother detectable moiety. Fluorophore moiety labels are preferred for usein the present invention because of their widespread availability andrelative ease of use.

In one preferred embodiment, the hybridized amplicons are detected byhybridization of a reporter species, such as a distinct target-specificoligonucleotide, that is labeled such that a detectable signal isproduced, either directly or in combined action with an additionalcomponent(s) of a signal-producing system. An example of a directlydetectable label is a fluorescent moiety that is present on thereporter, including, but not limited to, bodipy dyes such as BodipyTexas Red and cyanine dyes such as Cy3 and Cy5. In a preferredembodiment in which the capture probe complementary to the housekeepinggene, or the control, is combined with each target-specific captureprobe and co-addressed to a given position on the microarray, thereporter for the said housekeeping gene is labeled with a fluorophoreemitting at one wavelength, while the reporters for the amplicons of thegenes of interest are labeled with a different fluorophore that emits atleast one other wavelength. Analysis of signal generated at eachwavelength allows detection of all species hybridized on the microarrayand subsequent normalization using the expression data from thehousekeeping gene.

In another preferred embodiment, detection of at least a portion of thehybridized amplicons of the genes of interest is accomplished byenzymatic reporting. In this variation, after electronic hybridization,the microarray is incubated with reagents that allow primer extension ofthe capture probe using the bound RNA or cDNA amplicon target(s) as atemplate. For RNA targets, the extension is performed using an enzymewith reverse transcriptase activity, e.g. Superscript® reversetranscriptase, that uses RNA-DNA hybrids, but not RNA-RNA or DNA-DNAhybrids, as a template. Utilization of such enzyme will reducenon-specific signal that otherwise may be produced from extension ofself-annealed regions of either capture probes or target molecules. ForcDNA targets, an enzyme with DNA polymerase activity is used. In eithercase, one or more of the nucleotides present in the reaction will belabeled. The nucleotide species may be either deoxynucleotide(s) ordideoxynucleotide(s) and may be labeled with any detectable moiety,usually a fluorescent moiety. Preferred fluorescent moieties for use inthe primer extension methods of the invention include cyanine dyes, e.g.Cy5 and Cy3, and other fluorescent dyes such as Bodipy Texas Red,rhodamine, fluorescein, and cumarin. Incorporation of the labelednucleotide(s) into the extended capture probe will allow target-specifichybridization of the amplicons to be detected as a fluorescent signal atthe individual test sites in the array. Other reagents included in thereaction include buffer medium for optimal enzymatic activity; suchmedium is commercially available and known to those of skill in the art.

In embodiments where the capture probe for the housekeeping target isco-addressed with target-specific capture probes, these two species mustbe differentiated at the reporting step. In one variation, a reporterspecific for the housekeeping gene is allowed to hybridize prior to,concurrent with, or after the primer extension of capture probesspecific for the genes of interest. The reporter is modified (e.g., byphosphoramidite chemistry or other means known in the art) at the 3′ endto prevent extension of the reporter species during the primer extensionreaction. The housekeeping gene-specific reporter is labeled such thatthe signal from the hybridized reporter can be distinguished from thesignal generated from the extended target-specific capture probes, e.g.the reporter is labeled with Cy3 and the labeled NTP(s) used in theprimer extension reaction is labeled with Cy5. In a preferred variation,the capture probe for the housekeeping gene is also modified at the 3′end, e.g. with an amino-blocking group, to prevent primer extension. Useof a blocked capture probe allows simultaneous hybridization of thereporter complementary to the housekeeping gene and primer extension ofthe target-specific capture probes, since the blocked capture probehybridized to the housekeeping gene will not be extended.

The hybridization patterns are analyzed after signal detection. Thesignal generated by the housekeeping gene, which is expressed atequivalent levels in the different samples tested, is used to normalizedifferences in total nucleic acid concentration, electronic transport,and electronic hybridization efficiencies between samples, and toaccount for micro-environmental variations between test sites.Differences in intensities in the target amplicon signals afternormalization is an indication of altered expression levels in theoriginal mRNA in the sample nucleic acids under the conditions examined.

Applications of the Present Methods

The methods of the present invention may be readily applied to a widevariety of gene expression experimental models for use in studying, forexample, disease and oncogenesis, physio-chemical cellular responses tostimuli, and cell growth and differentiation. The disclosed methods areideal for these applications because of their speed, reproducibility,and flexibility as to the number, kind, and concentration of gene probesused in the hybridization experiment. As generally outlined below, thedisclosed methods can greatly increase the ease and rapidity of commonexpression experiments.

For example, the methods of the present invention may easily be used totitrate the amount of amplified mRNA present in a sample. As illustratedin FIG. 5, a sample of amplified nucleic acids may be serially diluted.Each dilution may then be specifically hybridized with a subset ofelectronically activated test sites on a NanoChip™ device. By producinga concentration curve from the serial dilution data, the originalconcentration of the amplified mRNA sequence in the sample may bedetermined. If a specific amount of control sequence is added to thesample prior to amplification for use as a control, this amplified mRNAconcentration may also be utilized to determine the original mRNAconcentration in the sample. Thus, the methods of the invention may beused to obtain absolute quantitative measurements, as well as relativequantitative measurements of gene expression.

The methods of the present invention may also be used advantageously tocompare, side by side, the expression levels of mRNA in a cell type thathas undergone two different physical or chemical stimuli. For instance,in Example 5, the expression levels of four genes in monocyte cellswhich were treated with LPS (lipopolysaccharide) or untreated (control)were examined. As shown in FIG. 6, a marked change in several genes wasobserved between the LPS treated monocytes and the untreated monocytes.Similar experiments may easily be devised by those of skill in the artfor screening potential chemical inducers or repressors of geneexpression for use in combinatorial-library high-throughput drugdiscovery, while monitoring the effects of the compound on non-targetgene expression pathways to minimize side effects.

Because of the flexibility of being able to perform multiple sampletests on the same electronically assisted hybridization device, thespecific format of an assay may also be easily changed to suit newdirections in a particular research project. For instance, a researchermay initially use a 100 test site NanoChip™ to screen 50 different genesin two samples from a cell line that has and has not been exposed to achemical agent. Upon identifying 5 genes of particular interest, theresearcher may then strip off the old probes from the streptavidinpermeation layer, reconfigure the 100 test sites with using just 5 ofthe original 50 probes, and verify the result with a larger sample setof 5 controls and 15 stimulated cell samples, using 3 groups of cellsstimulated with different concentrations of the chemical agent. Once anapparent rough critical concentration of chemical agent has beenidentified, four cell samples may be stimulated with concentrations ofthe agent centered around the rough critical concentration. The amountof amplified mRNA produced at each concentration may then be determinedfrom four serial dilutions of each sample, utilizing the NanoChip™ asconfigured for screening the 20 samples above. As each screening stepmay be accomplished in 10 minutes to an hour, the entire project may becompleted in a manner of days utilizing the methods of the presentinvention, depending on the stimulation period allotted for the cellsamples

The following examples are offered to further illustrate the variousaspects of the present invention, and are not meant to limit theinvention in any fashion. Based on these examples, and the precedingdiscussion of the embodiments and uses of the invention, severalvariations of the invention will become apparent to one of ordinaryskill in the art. Such self-evident alterations are also considered tobe within the scope of the present invention.

EXAMPLES Example 1

General Protocol for Gene Expression Monitoring with Shortened Amplicons

A. cDNA synthesis.

Total RNA (5 μg) or poly(A⁺) RNA(0.5 μg) was used as template in cDNAsynthesis reactions with Superscript™ II RNase H⁻ Reverse Transcriptase(Life Technologies, Rockville, Md.) per manufacturer's instructions,with the inclusion of 10 U RNase Inhibitor (Roche Diagnostics, Mannheim,Germany). Synthesis was primed with either 500 ng oligo(dT)₁₂₋₁₈ (LifeTechnologies), or with oligo(dT)₁₇V (where V=A, G, or C), or with anoligo(dT₁₈T_(mod)N₃) containing a non-replicable 1,3-propane diol moietyon the fourth base from the 3′ end (U.S. Pat. No. 6027923). When anexogenous target gene was used as a control, 100-500 pg poly(A⁺) RNA forthe target was included in this initial cDNA synthesis reaction.

B. Linear amplification using DNA polymerase.

One-fourth to one-half of the cDNA synthesis reactions was used inVent_(R)®(exo³¹) DNA polymerase (New England Biolabs, Beverly, Mass.)mediated primer extension reactions. 1×Thermopol Buffer (New EnglandBiolabs), 200 nM each dNTP (Roche Molecular Biochemicals, Indianapolis,Ind.), 50 nM each biotinylated chimeric T7-target specificoligonucleotide [See Table 2], and 0.04 U/ml reaction Vent_(R)(exo⁻) DNApolymerase were used. Final Mg⁺⁺ concentration was generally 2.3 mM, butwas occasionally adjusted to as much as 4.6 mM. Chimeric primers for oneto nine specific targets were added, in addition to a chimericoligonucleotide specific for the internal (exogenous or endogenous)control. Samples were denatured at 95° C. 10 min, then cycled 30-50times at (95° C. 30 sec, 50-60° C. 60 sec, 72° C. 60 sec). After thelast cycle, samples were held at 72° C. for 5 min. 50 ng random hexamerprimers. (Life Technologies) or 200 nM target-specificoligonucleotide(s) were added and annealed to the amplified targets at42° C., 10 min. Annealed primers were extended by increasing thetemperature to 70° C., 10 min.

C. Binding of amplified targets to streptavidin-coated magnetic beadsand in vitro transcription reactions.

Amplified targets were isolated from other components of theamplification reaction mixture by binding to streptavidin-coatedmagnetic beads (Dynal®, Oslo, Norway) 30-60 min at 37° C. withoccasional agitation. Beads were then isolated on a magnetic separator(Dynal) and the bound target was used as template for in vitrotranscription reactions using the T7 MegaShortScript in vitrotranscription kit (Ambion, Inc., Austin, Tex.). Reactions were incubatedas per manufacturer's instructions 2-4 hr at 37° C. Transcribed RNA waspurified from excess NTPs by isolation with Chromaspin-30 columns(Clontech, Palo Alto, Calif.) as per manufacturer's instructions. TheRNA targets were used directly for analysis on microelectronic arrays,or used as template in a second cDNA synthesis reaction as describedbelow.

D. Second round cDNA synthesis

20 μl of in vitro transcribed RNA was used as template in a second cDNAsynthesis reaction. 50 ng random hexamer primers (Life Technologies)(sometimes used in multiplex analysis amplification) or 200 nMtarget-specific oligonucleotide(s) were annealed to RNA targets at 70°C. 10 min. 1×First strand buffer, 10 mM DTT, and 0.5 mM dNTPs (RocheMolecular Biochemicals, Indianapolis, Ind.) were added; samples wereincubated at 42° C. 2 min prior to addition of 300 U Superscript™ IIRNase H⁻ reverse transcriptase (Life Technologies). Reactions wereincubated at 42° C. 50-75 min. 1 μg RNaseA (Ambion, Inc.) and 1 U RnaseH(Life Technologies) were added per reaction and samples were incubatedat 37° C. 10 min. Samples were then desalted on either Chromaspin-30columns (Clontech) or Bio-Spin 6 columns (Bio-Rad Laboratories,Hercules, Calif.) and used on microelectronic arrays. N.B. Whenrestriction digestion is done, we use RNA target, we don't convert itinto cDNA. The choice of random hexamer v. gene specific oligo for cDNAsynthesis is based on the advantage random hexamers provide inmultiplexing experiments weighed against the possible reduction inamount of detectable cDNA generated.

E. Electronic capture addressing and target hybridization.

500 nM target-specific biotinylated capture oligonucleotide in 50 mMhistidine was electronically addressed to specific sites on theNanoChip™, using the Nanogen Molecular Biology Workstation. Probes weretransported using 2.0 V constant voltage for 1 minute; typically, 2-10pads were addressed simultaneously. Unbound oligonucleotides wereremoved by washing with 50 mM histidine. When an internal control wasincluded within the sample, 500 nM capture for this species wasco-addressed with target-specific capture oligonucleotide; this internalcontrol capture is blocked at the 3′ end with an amino modifier toprevent extension in the subsequent reporting reaction.

RNA or cDNA targets in 50 mM histidine were heat-denatured by incubationat 95° C. 5 min and quick-chilled on ice. Targets were electronicallyhybridized to specific capture oligonucleotides using 2.0V for 2-3 min;typically, 2-10 sites on the array were addressed simultaneously.Unhybridized nucleic acids were removed by washing with 50 mM histidine.

F. Reporting

The internal control target is detected by hybridization with 0.5-1 μM3′ Cy3-labeled oligonucleotide that binds directly adjacent to thecapture oligonucleotide; this reporter oligonucleotide can be includedin the enzymatic reporting mixture. Targets of interest that arehybridized to capture oligonucleotides are detected by extension of thecapture probes in the presence of Cy5-labeled dCTP. For RNA targets,after the final hybridization reaction and washing was completed, theNanoChip™ was incubated with 1×First strand buffer (Life Technologies);10 mM DTT (Life Technologies); 0.25 mM each dATP, cGTP, and dTTP (RocheMolecular Biochemicals, Indianapolis, Ind.); 0.125 mM Cy5-dCTP (AmershamPharmacia Biotech, Buckinhamshire, England); and 200 U Superscript™ IIRNase H⁻ reverse transcriptase (Life Technologies) for 10 min at 37° C.For cDNA targets, the NanoChip was incubated in 50 mM NaPO₄ for 10 minat 37° C. prior to enzymatic extension of the captureoligonucleotide(s). Reporting of hybridized cDNA targets was performedas described for RNA targets, except that 1×Second strand buffer (LifeTechnologies) and 20 U DNA polymerase I (Life Technologies) were used inplace of First strand buffer and reverse transcriptase. Following theenzymatic extension reaction, NanoChip™ were washed with 50 mM NaPO₄.Due to the high salt content of the enzymatic reporting mixture, theincluded Cy-3 labeled internal control probe hybridizes to thehousekeeping sequence fairly rapidly at the 37° C. incubationtemperature. Fluorescence from the Cy3-labeled internal control reporterand from the incorporated Cy5-dCTP was detected on the Nanogen MolecularBiology Workstation.

For quantitation of targets, the signal from the Cy3 labeled internal(exogenous or endogenous) control gene, the expression of which is notexpected to change under the conditions being examined, is equalized,and a normalization factor is thus obtained. Target-specific signal (Cy5) is then adjusted by this normalization factor. The amount of changein target gene expression level under the experimental condition maythen be determined by comparing these adjustedhybridization/fluorescence values.

Example 2

Enzymatic-mediated Shortening of Targets

cDNA synthesis and amplification were as described above. FollowingVent_(R)®(exo⁻) DNA polymerase-mediated amplification and fill-inreactions, the targets were enzymatically shortened by digestion withthe type IIs restriction endonuclease BpmI, utilizing a BpmI recognitionsite in the endogenous sequence of IL6, TGFβ1, and p53 and chimericprimer altered sites in COX1 and GAPDH, as described in Example 4.Digestion was performed at 37° C. for 1.5-2 h. Streptavidin-coatedmagnetic beads were then added and incubation was continued at 37° C.for an additional 30-60 min. The bead-bound targets were used in invitro transcription reactions as described above, and RNA targets wereanalyzed on NanoChip™ microarrays.

Example 3

Re-use of Targets Bound to Streptavidin-coated Magnetic Beads forMultiple in vitro Transcription Reactions.

After the initial cDNA synthesis reaction and Vent_(R)® (exo⁻) DNApolymerase mediated amplification, as described above, double strandedtarget(s) are immobilized on streptavidin-coated magnetic beads via thebiotin moiety introduced by the chimeric T7 RNA polymerase recognitionsite-gene specific oligonucleotide. These targets are then used astemplates in in vitro transcription reactions, as described above.Following in vitro transcription, the bead-bound target can be stored at4° C. and re-used in subsequent in vitro transcription reactions. Thisaspect of the protocol allows re-analysis of targets of interest from agiven sample pool without having to obtain more of the initial cellularRNA.

FIG. 3 demonstrates the results obtained when a single pool ofbead-bound targets is utilized in three separate rounds of in vitrotranscription. Note that, unexpectedly, the amount of transcriptproduced decreases only slightly between the first and third rounds oftranscription, and the decrease in the amount of transcript is notsignificant between transcription rounds. Several additional rounds ofin vitro transcription are possible, with a minimal iterative decreasein the amount of transcript produced.

Example 4

Demonstration of the Effect of Amplicon Size on Hybridization Efficiencyin Electronic and Passive Hybridization Systems

Cellular messenger RNA was isolated from cultured U937 monocyte cells(see Example 5) and reverse transcribed into cDNA with oligo(dT)₁₂₋₁₇.The hybridization of COX1, GAPDH, IL6, TGFβ1, and p53 amplicons ofapproximately 50, 100, 250, and 500 bases in size were determinedutilizing both electronic (as described above) and passive hybridizationprocedures (outlined below), using a TPOX control for normalization ofthe hybridization values. One primer for each target contained the T7RNA polymerase recognition site upstream of gene specific sequence;these primers were designed to incorporate a BpmI site at the 3′ end.The other primers were designed to generate products that were 100 bp,250 bp, or 500 bp in length. A portion of the 100 bp amplified targetwas digested with BpmI to generate 50 bp fragments. After amplification,similarly sized targets were pooled and used as template in in vitrotranscription reactions with the T7 MegaScript or MegaShortScript kits(Ambion, Inc.). The different pools of RNA targets were then hybridizedpassively or electronically to microarray sites co-addressed withtarget-specific and control (TPOX)-specific capture oligonucleotides.Hybridized targets were detected by enzymatic extension of thehybridized capture oligonucleotides in the presence of Cy5-dCTP. In thecase of the internal control TPOX, this reporter was labeled with Cy3;.

A) Exponential amplification of target sequences

For exponential amplification of cDNA, one-tenth (2 μl) of a standardcDNA synthesis reaction was utilized. Amplification was performed in thepresence of 1×PCR buffer II (Perkin Elmer, Foster City, Calif.); 0.2 μMeach primer; 200 μM dNTP mix; and 2.5 U AmpliTaq Gold (Perkin Elmer).cDNA was denatured at 95° C. 10 minutes, then amplified by cycling (95°C. 30 sec; 50-60° C. 60 sec; 72° C. 120 sec). Amplification reactionswere generally performed for 25-30 cycles. The annealing temperature waschosen to be 5° C. lower than the Tm of the primers.

B) Passive hybridization of RNA targets

10 nM RNA targets were passively hybridized to capture-loadedmicroarrays by incubation in 4×SSC, 1×Denhard's , and 100 μg yeast tRNA.Hybridization was performed by placing the microarray on a moist pieceof filter paper in a small petri dish. The dish was sealed with Parafilm(American National Can, Neenah, Wis.) and the microarray was incubated14-16 hr at 37° C. The hybridization solution was subsequently removedand the unbound target removed by washing with 50 mM NaPO₄.

C) Passive hybridization of labeled reporter oligonucleotides

In the case where fluorescently labeled reporter oligonucleotidesexclusively are used to detect target species on the microarray, 1 μMeach reporter species is passively hybridized to the immobilized targetin 50 mM NaPO₄/500 mM NaCl. This hybridization reaction is performed at23° C. for 5 min; unbound reporters are then removed by washing with 50mM NaPO₄ prior to imaging of the microarray.

The hybridizations utilized to generate the electronic hybridizationdata are shown in FIG. 4.

TABLE 1 Effect of target size on hybridization efficiency Target Gene:50 nt 100 nt 250 nt 500 nt ELECTRONIC HYBRIDIZATION: (After 2-3 minutes)COX2 36.6 45.8 21.0 4.62 GAPDH 77.2 95.0 39.5 2.22 IL6 46.9 69.2 47.349.2 TGFβ 206 187 131 16.9 p53 17.5 19.4 1.65 ND PASSIVE HYBRIDIZATION:(After 14-16 hours) COX1 34.4 28.0 6.06 5.51 GAPDH 68.1 75.0 6.89 2.20IL6 71.3 26.4 12.9 6.91 TGFβ1 150 150 38.2 6.86 p53 23.0 14.6 6.17 6.76

These numbers represent fluorescence units

As can be seen from the above data, the length of the amplicons used inthe hybridization procedure had a marked effect on hybridizationefficiency.

Example 5

Demonstration of Gene Expression Monitoring in LPS Stimulated MonocyteCells

The monocytic cell line U937 was cultured to 4×10⁷ cells/75 cm² flask inRPMI media (American Type Culture Collection, Manassas Va.) containing10% Fetal Bovine Serum (American Type Culture Collection) and 200units/g/ml penicillin/streptomycin (Life Technologies) in two or moreflasks. Cells were collected and resuspended in eitherRPMI/FBS/antibiotics or in RPMI/FBS/antibiotics+phorbol myristate acid(Life Technologies) at a final concentration of 10 ng/ml. 72 h after PMAtreatment, cells were collected and resuspended in RPMI media containingFBS and antibiotics; PMA-treated cells were induced by treatment with500 ng/ml lipopolysaccharide (Sigma, St. Louis, Mo.). Cells wereharvested 6 h after addition of LPS. Uninduced cells that had beenmaintained in RPMI/FBS/antibiotic medium were also collected(“Uninduced”). Untreated and treated cells were snap frozen in liquidnitrogen until needed for mRNA isolation.

mRNA isolation was performed with the Poly(A) Pure Isolation kit(Ambion, Inc.). Cellular poly(A⁺) RNA was isolated and cDNA wasgenerated using the propane diol-modified oligo dT primer. Limitedamplification reactions were performed in duplex reactions as follows:the biotinylated chimeric T7-gene specific oligonucleotides forInterleukin I beta (IL1) and Transforming Growth Factor beta-2 (TGFβ2)were used in one amplification reaction; and the biotinylated chimericT7-gene specific oligonucleotides for Interleukin 6 (IL6) and c-jun wereused in a second amplification reaction. Single stranded targets werefilled in by addition of gene specific oligonucleotides and used in invitro transcription reactions. The RNA generated was used as templatefor second cDNA syntheses. Electronic capture addressing, targethybridization, and enzymatic extension with DNA polymerase were carriedout as described in Example 1.

The hybridization results of the experiment are shown in FIG. 6, andgraphically represented in FIG. 7. cNS represents a non-specificbiotinylated oligonucleotide that does not contain sequencecomplimentary to any of the amplified targets. As shown, the addition ofLPS to the monocyte cell culture provoked dramatically increased mRNAexpression for IL1, moderately increased expression of TGFβ2 and c-junmessage, and minimally increased expression of IL6. Thus, asillustrated, altered expression of genes between two sample cellpopulations can be easily monitored using the methods of the presentinvention.

Example 6

Demonstration of Serial Dilution and Quantitative Methods Utilizing theNanoChip™ Electronic Hybridization Device

A) Detection of Serially Diluted Short RNA Targets on the NanoChip™

The β-la target gene was amplified from plasmid DNA using chimeric genespecific oligonucleotides, as described above. The 5′ primer (pT7Amp.s1) contains a T7 RNA polymerase recognition sequence upstream ofgene specific sequence; the 3′ primer contains a poly(dT) tractdownstream of gene specific sequence. This PCR product, representing thefull-length β-la gene, was used as template in in vitro transcriptionreactions with the T7 Megascript kit per manufacturer's instructions(Ambion, Inc.). The target was reverse transcribed to generate cDNA, andthe cDNA product was used in a Vent_(R)® (exo⁻) DNA polymerase-mediatedamplification reaction with a second, internal chimeric T7 RNApolymerase recognition site/gene-specific oligonucleotide(pT7AmpBpm.s1). The amplified target was enzymatically digested withBpmI and used in a second in vitro transcription reaction using the T7MegaShortScript kit (Ambion, Inc.). The resulting short RNA target wasserially diluted as indicated and electronically hybridized to a β-laspecific capture oligonucleotide. The electronic hybridization data isshown in FIG. 5a.

B) Detection of Exogenous Target in a Pool of Cellular RNA

The full length β-la target was generated as described above. This RNAwas added in defined amounts (0 to 800 pg) to 250 ng cellular messengerRNA. The entire pool of RNA was then reverse transcribed into cDNA, andthe β-la target was amplified in a Vent® (exo⁻) DNA polymerase-mediatedamplification reaction. The amplified target was enzymatically digestedwith BpmI and used in a second in vitro transcription reaction using theT7 MegaShortScript kit (Ambion, Inc.). The resulting short RNA targetwas electronically hybridized to a β-la specific captureoligonucleotide. The hybridization data is shown in FIG. 5b, andgraphically represented in FIG. 5c.

TABLE 2 Oligonucleotides Used in the Above Experimental Procedures,Organized by Target Gene Primer name and Target Gene description Primersequence Angiotensinogen bAt7AngBpm.s1- BiotinTAATACGACTCACTATAGGbiotinylated chimeric T7 GAGACACAGAACTGGATGTTGC promoter/gene specificTGCTGGAG [SEQ.ID NO.1] oligonucleotide Angiotensinogen cAng.a1-capturefor BiotinCATGAACCTGTCAATCUCT RNA [SEQ. ID NO.2] AngiotensinogenpAng.a1-3′ gene GGAAGGTGCCCATGCCAGAGA specific primer [SEQ. ID NO.3]Cathepsin G bAt7CathBpm.s1 - BiotinTAATACGACTCACTATAGG biotinylatedchimeric T7 GAGAGCTGCCTTCAAGGGGGAT promoter/gene specific TCTGGAG [SEQ.ID NO.4] oligonucleotide Cathepsin G pCath.a1-3′ geneAGCTTCTCATTGTTGTCCTTATC specific primer C [SEQ. ID NO.5] Cathepsin GcCath.a1-capture for BiotinTGTTACACAGCAGGGGGC RNA CT [SEQ. ID NO.6]c-jun bT7jun.s1-biotinylated BiotinTAATACGACTCACTATAGG chimeric T7GAGACGGCCAACATGCTCAGGG promoter/gene specific AACAGGT [SEQ. ID NO.7]oligonucleotide c-jun cjun.s1-capture for BiotinCAAACATTTTGAAGAGAGA cDNACCGTCG [SEQ. ID NO.8] c-jun pjun.a1-3′ gene specificTTTTTCTTCGTTGCCCCTCAGCC primer [SEQ. ID NO.9] COX1 pcox1.as.3-geneTGCCCAGGATTGATTCACAGG specific primer for [SEQ. ID NO.10] generation of500 bp fragment COX1 pcox1.as.2-gene AGGCCAGAAGGAATGATGGG specificprimer for [SEQ. ID NO.11] generation of 250 bp fragment COX1pcox1.as.1-gene CTAAGCCCAAAGTGTGGATC specific primer for [SEQ. ID NO.12]generation of 100 bp fragment COX1 T7.cox1.s-chimericGAAATTAATACGACTCACTATAG T7/gene specific GGAGAACCCTTTTCTCAGGACCToligonucleotide CTGGAGG [SEQ. ID NO.13] COX1 cCOX1bpm.a1-captureBiotinACAGAGGTCCTGAGAAAAG for RNA GGTCT [SEQ. ID NO.14] COX2bAt7COX2bpm.s2- BiotinTAATACGACTCACTATAGG biotinylated chimeric T7GAGACTATGAATCATTTGAAGAA promoter/gene specific CTTACTGGAG [SEQ. IDNO.15] oligonucleotide COX2 cCOX2.a2-capture forBiotinCTGCAGACATTTCCTTTTCT RNA [SEQ. ID NO.16] COX2 pCOX2.a2-3′ geneGCATCTGGCCGAGGCTTTTCTAC specific primer [SEQ.ID NO.17] GAPDHpGAPs.2-3′ gene specific GTTCGACAGTCAGCCGCATCTTC primer for generationof [SEQ. ID NO.18] 500 bp fragment GAPDH pGAPs.3-gene specificTGATGCCCCCATGTTCGTCATGG primer for generation of [SEQ. ID NO.19] 100 bpfragment GAPDH pGAPs.4-gene specific CTTCCAGGAGCGAGATCCCTCC primer forgeneration of [SEQ. ID NO.20] 250 bp fragment GAPDH T7GAPbpm.a1-chimericGTAATACGACTCACTATAGGGCG T7/gene specific GGGTGCTAAGCAGTTGGTGGTGoligonucleotide CTGGAG [SEQ. ID NO.21] GAPDH cGAPbpm.s1-captureBiotinCAGCCTCAAGATCATCAGC for aRNA AATGCCT [SEQ. ID NO.22] GAPDHbAt7GAPbpm.s2- BiotinTAATACGACTCACTATAGG biotinylated chimeric T7GAGACTCAAGGGCATCCTGGGC promoter/gene specific TACACTGGAGCAC [SEQ. ID NO.oligonucleotide 23] GAPDH pGAPDH.a6-3′ gene GAGGTCCACCACCCTGTTGCTGspecific primer TAG [SEQ. ID NO.24] GAPDH cGAPbpm.a2-captureBiotinGTTGAAGTCAGAGGAGACC for RNA ACCTGGTGCT [SEQ. ID NO.25] HMG-17bAt7HMG17Bpm.s1- BiotinTAATACGACTCACTATAGG biotinylated chimeric T7GAGAGGAATAACCCTGCAGAAA promoter/gene specific CTGGAG [SEQ. ID NO.26]oligonucleotide HMG-17 pHMG17.a1-3′ gene CCCTTCCCCCAAAAACAACAATGspecific primer A [SEQ. ID NO.27] HMG-17 cHMG17.al-capture forBiotinCCTGGTCTGTTTTGGCATC RNA T [SEQ. ID NO.28] Interleukin 6pIL6s.4-gene specific ATTCTGCCCTCGAGCCCACCGG primer for generation of G[SEQ. ID NO.29] 500 bp fragment Interleukin 6 pIL6S.3-gene specificCAAACAAATTCGGTACATCCTCG primer for generation of [SEQ. ID NO.30] 250bpfragment Interleukin 6 pIL6S.2-gene specific TGGATTCAATGAGGAGACTTGCCprimer for generation of [SEQ. ID NO.31] 100 bp fragment Interleukin 6T7IL6bpm.al-chimeric GTAATACGACTCACTATAGGGCG T7/gene specificCCTCACTACTCTCAAATCTGTTC oligonucleotide TGGAG [SEQ. ID NO.32]Interleukin 6 cIL6bpm.s1-capture for BiotinGGAGTTTGAGGTATACCTA aRNAGAGTACCT [SEQ. ID NO.33] Interleukin 6 bT7IL6.s1-biotinylatedBiotinTAATACGACTCACTATAGG chimeric T7 GAGACCTGAGGGCTCTTCGGCApromoter/gene specific AATGTAG [SEQ. ID NO.34] oligonucleotideInterleukin 6 cIL6.s1-capture for BiotinAATGGGCATTCCTTCTTCT cDNA GGTCAG[SEQ. ID NO.35] Interleukin 6 pII6.a1-3′ gene specificGAACAACATAAGTTCTGTGCCCA primer GTG [SEQ. ID NO.36] Interleukin 1 betabT7lL1.s1-biotinylated BiotinTAATACGACTCACTATAGG chimeric T7GAGACAGAAAACATGCCCGTCTT promoter/gene specific CCTGG [SEQ. ID NO.37]oligonucleotide Interleukin 1 beta cIL1.s1-capture forBiotinGCGGCCAGGATATAACTGA cDNA CTTCAC [SEQ. ID NO.38] Interleukin 1 betapII1.a1-3′ gene specific TCCACATTCAGCACAGGACTCTC primer TG [SEQ. IDNO.39] LD78 bAt7LD78Bpm.s1- BiotinTAATACGACTCACTATAGG biotinylatedchimeric T7 GAGAAGTGACCTAGAGCTGAGT promoter/gene specific GCCTGGAG [SEQ.ID NO.40] oligonucleotide LD78 pLD78.al-3′ gene CTCTCAGAGCAAACAATCACAAAspecific primer CACAC [SEQ. ID NO.41] LD78 cLD78.a1-capture forBiotinTCGAAGCTTCTGGACCCCT RNA [SEQ. ID NO.42] Osteopontin bAt7OstBpm.s1-BiotinTAATACGACTCACTATAGG biotinylated chimeric T7GAGAGAGGTGATAGTGTGGTTT promoter/gene specific ATGGACTGGAG [SEQ. IDNO.43) oligonucleotide Qsteopontin pOst.a1-3′ gene specificCAACGGGGATGGCCTTGTATGC primer [SEQ. ID NO.44] OsteopontincOst.a1-capture for BiotinAACTTCTTAGATTTTGACCT RNA [SEQ. ID NO.45] p53pp53s.3-gene specific ACAGAAACACTTTTCGACATAG primer for generation of[SEQ. ID NO.46] 500 bp fragment p53 pp53.s1-gene specificAAAGGGGAGCCTCACCACGAGC primer for generation of [SEQ. ID NO.47] 250 bpfragment p53 pp53.s1-gene specific CGTGAGCGCTTCGAGATGTTCC primer forgeneration of [SEQ. ID NO.48] 100 bp fragment p53 T7p53bpm.a1-chimericGTAATACGACTCACTATAGGGCG T7lgene specific ACCCTTTTTGGACTTCAGGTGGColigonucleotide TGGAG [SEQ. ID NO.49] p53 cp53bpm.s1-capture forBiotinGAGCCAGGGGGGAGCAGG aRNA GCTCACT [SEQ. ID NO.50] TGFβ1pTGFb1S.3-gene GGGATAACACACTGCAAGTGGA specific primer for C [SEQ. IDNO.51] generation of 500 bp fragment TGFβ1 pTGFb1s.2-geneCCACGAGCCCAAGGGCTACCAT specific primer for GC [SEQ. ID NO.52] generationof 250 bp fragment TGFβ1 pTGFb1.s1-gene CGCTGGAGCCGCTGCCCATCGT specificprimer for GTA [SEQ. ID NO.53] generation of 100 bp fragment TGFβ1T7TGFb1bpm.al- GTAATACGACTCACTATAGGGCG chimeric T7/gene specificGGCGGGACCTCAGCTGCACTTG oligonucleotide CTGGAG [SEQ. ID NO.54] TGFβ1cTGFb1bpm.s1-capture BiotinCAGCTGTCCAACATGATCG for aRNA TGCGCT [SEQ. IDNO.55] TGFβ2 bT7TGFb.s1- BiotinTAATACGACTCACTATAGG biotinylated chimericT7 GAGACTCTGCCTCCTCCTGCCT promoter/gene specific GTCTGC [SEQ. ID NO.56]oligonucleotide TGFβ2 cTGFb.s1-capture for BiotinCGGCATCAAGGCACAGGG cDNAGACCAGT [SEQ. ID NO.57] TGFβ2 pTGFb.a1-3′ gene CTTCAACAGTGCCCAAGGTGCTspecific primer CAA [SEQ. ID NO.58) TPOX TPOX9C-biotinylatedBiotinTTAGGGAACCCTCACTGAA synthetic target TGAATGAATGAATGAATGAATGAATGAATG [SEQ. ID NO.59] TPOX TPOXcapcomp-Cy3 CATTCATTCATTCAGTGAGGGTTlabeled reporter for CC [SEQ. ID NO.60] TPOX9C Vimentin bAt7VimBpm.s2-BiotinTAATACGACTCACTATAGG biotinylated chimeric T7GAGACATCGACAAGGTGCGCTT promoter/gene specific CCTGGAG [SEQ. ID NO.61]oligonucleotide Vimentin pVim.a1-3′ gene CGCGGGCTTTGTCGTTGGTTAG specificprimer [SEQ. ID NO.62] Vimentin cVim.a2-capture forBiotinCAGGATCTTATTCTGCTGC RNA T [SEQ. ID NO.63] β-ActinbAt7Actin.s-biotinylated BiotinTAATACGACTCACTATAGG chimeric T7GAGACCCCTTTTTGTCCCCCAAC promoter/gene specific TGGAGA [SEQ. ID NO.64]oligonucleotide β-Actin cActin.a-capture for BiotinCCAAAAGCCTTCATACATCRNA T [SEQ. ID NO.65] β-Actin pbAa.4-3′ gene specificAAGGTGTGCACTTTTATTCAACT primer GGTCTCAAG [SEQ. ID NO.66] β-IapT7AmpBpm.s1- TAATACGACTCACTATAGGCTGG chimeric T7/gene specificCTGGTTTATTGCTGATAAATCTG oligonucleotide for GAG [SEQ. ID NO.67]generation of short RNA β-Ia pAmp.a2-3′ primer withT₃₀CCAATGCTTAATCAGTGAGGC poly(dT) tract ACCTATCTC [SEQ. ID NO.68] β-IacAmp.a1-capture for biotin- RNA CGAGACCCACGCTCACCGGCT [SEQ. ID NO.69]β-Ia pT7Amp.s1-chimeric TAATACGACTCACTATAGGGCAC T7/gene specificCCAGAAACGCTGGTGAAAGTAA oligonucleotide for AAG (SEQ. ID NO.70]generation of full-length gene β- bAt7Throm.s1-BiotinTAATACGACTCACTATAGG Thromboglobulin- biotinylated chimeric T7GAGAGGAAAACTGGGTGCAGAG like protein gene promoter/gene specificGGTTCTGGAG [SEQ. ID NO.71] oligonucleotide β- pbThrom.a1-3′ geneGGCAACCCTACAACAGACCCAC Thromboglobulin- specific primer AC [SEQ. IDNO.72] like protein gene β- cbThrom.a1-capture forBiotinAGCCCTCTTCAAAAACTTCT Thromboglobulin- RNA [SEQ. ID NO.73] likeprotein gene

73 1 49 DNA Homo sapiens modified_base (1)..(1) Biotinylated 1taatacgact cactataggg agacacagaa ctggatgttg ctgctggag 49 2 20 DNA Homosapiens modified_base (1)..(1) Biotinylated 2 catgaacctg tcaatcttct 20 321 DNA Homo sapiens 3 ggaaggtgcc catgccagag a 21 4 48 DNA Homo sapiensmodified_base (1)..(1) Biotinylated 4 taatacgact cactataggg agagctgccttcaaggggga ttctggag 48 5 24 DNA Homo sapiens 5 agcttctcat tgttgtccttatcc 24 6 20 DNA Homo sapiens modified_base (1)..(1) Biotinylated 6tgttacacag cagggggcct 20 7 48 DNA Homo sapiens modified_base (1)..(1)Biotinylated 7 taatacgact cactataggg agacggccaa catgctcagg gaacaggt 48 825 DNA Homo sapiens modified_base (1)..(1) Biotinylated 8 caaacattttgaagagagac cgtcg 25 9 23 DNA Homo sapiens 9 tttttcttcg ttgcccctca gcc 2310 21 DNA Homo sapiens 10 tgcccaggat tgattcacag g 21 11 20 DNA Homosapiens 11 aggccagaag gaatgatggg 20 12 20 DNA Homo sapiens 12 ctaagcccaaagtgtggatc 20 13 53 DNA Homo sapiens 13 gaaattaata cgactcacta tagggagaacccttttctca ggacctctgg agg 53 14 24 DNA Homo sapiens modified_base(1)..(1) Biotinylated 14 acagaggtcc tgagaaaagg gtct 24 15 52 DNA Homosapiens modified_base (1)..(1) Biotinylated 15 taatacgact cactatagggagactatgaa tcatttgaag aacttactgg ag 52 16 20 DNA Homo sapiensmodified_base (1)..(1) Biotinylated 16 ctgcagacat ttccttttct 20 17 23DNA Homo sapiens 17 gcatctggcc gaggcttttc tac 23 18 23 DNA Homo sapiens18 gttcgacagt cagccgcatc ttc 23 19 23 DNA Homo sapiens 19 tgatgcccccatgttcgtca tgg 23 20 22 DNA Homo sapiens 20 cttccaggag cgagatccct cc 2221 51 DNA Homo sapiens 21 gtaatacgac tcactatagg gcggggtgct aagcagttggtggtgctgga g 51 22 26 DNA Homo sapiens modified_base (1)..(1)Biotinylated 22 cagcctcaag atcatcagca atgcct 26 23 54 DNA Homo sapiensmodified_base (1)..(1) Biotinylated 23 taatacgact cactataggg agactcaagggcatcctggg ctacactgga gcac 54 24 25 DNA Homo sapiens 24 gaggtccaccaccctgttgc tgtag 25 25 29 DNA Homo sapiens modified_base (1)..(1)Biotinylated 25 gttgaagtca gaggagacca cctggtgct 29 26 47 DNA Homosapiens 26 taatacgact cactataggg agaggaataa ccctgcagaa actggag 47 27 24DNA Homo sapiens 27 cccttccccc aaaaacaaca atga 24 28 20 DNA Homo sapiensmodified_base (1)..(1) Biotinylated 28 cctggtctgt tttggcatct 20 29 23DNA Homo sapiens 29 attctgccct cgagcccacc ggg 23 30 23 DNA Homo sapiens30 caaacaaatt cggtacatcc tcg 23 31 23 DNA Homo sapiens 31 tggattcaatgaggagactt gcc 23 32 51 DNA Homo sapiens 32 gtaatacgac tcactatagggcgcctcact actctcaaat ctgttctgga g 51 33 27 DNA Homo sapiensmodified_base (1)..(1) Biotinylated 33 ggagtttgag gtatacctag agtacct 2734 48 DNA Homo sapiens modified_base (1)..(1) Biotinylated 34 taatacgactcactataggg agacctgagg gctcttcggc aaatgtag 48 35 25 DNA Homo sapiensmodified_base (1)..(1) Biotinylated 35 aatgggcatt ccttcttctg gtcag 25 3626 DNA Homo sapiens 36 gaacaacata agttctgtgc ccagtg 26 37 47 DNA Homosapiens modified_base (1)..(1) Biotinylated 37 taatacgact cactatagggagacagaaaa catgcccgtc ttcctgg 47 38 25 DNA Homo sapiens modified_base(1)..(1) Biotinylated 38 gcggccagga tataactgac ttcac 25 39 25 DNA Homosapiens 39 tccacattca gcacaggact ctctg 25 40 49 DNA Homo sapiensmodified_base (1)..(1) Biotinylated 40 taatacgact cactataggg agaagtgacctagagctgag tgcctggag 49 41 28 DNA Homo sapiens 41 ctctcagagc aaacaatcacaaacacac 28 42 19 DNA Homo sapiens modified_base (1)..(1) Biotinylated42 tcgaagcttc tggacccct 19 43 52 DNA Homo sapiens modified_base (1)..(1)Biotinylated 43 taatacgact cactataggg agagaggtga tagtgtggtt tatggactggag 52 44 22 DNA Homo sapiens 44 caacggggat ggccttgtat gc 22 45 20 DNAHomo sapiens modified_base (1)..(1) Biotinylated 45 aacttcttagattttgacct 20 46 22 DNA Homo sapiens 46 acagaaacac ttttcgacat ag 22 4722 DNA Homo sapiens 47 aaaggggagc ctcaccacga gc 22 48 22 DNA Homosapiens 48 cgtgagcgct tcgagatgtt cc 22 49 51 DNA Homo sapiens 49gtaatacgac tcactatagg gcgacccttt ttggacttca ggtggctgga g 51 50 25 DNAHomo sapiens modified_base (1)..(1) Biotinylated 50 gagccaggggggagcagggc tcact 25 51 23 DNA Homo sapiens 51 gggataacac actgcaagtg gac23 52 24 DNA Homo sapiens 52 ccacgagccc aagggctacc atgc 24 53 25 DNAHomo sapiens 53 cgctggagcc gctgcccatc gtgta 25 54 51 DNA Homo sapiens 54gtaatacgac tcactatagg gcgggcggga cctcagctgc acttgctgga g 51 55 25 DNAHomo sapiens modified_base (1)..(1) Biotinylated 55 cagctgtccaacatgatcgt gcgct 25 56 47 DNA Homo sapiens modified_base (1)..(1)Biotinylated 56 taatacgact cactataggg agactctgcc tcctcctgcc tgtctgc 4757 25 DNA Homo sapiens modified_base (1)..(1) Biotinylated 57 cggcatcaaggcacagggga ccagt 25 58 25 DNA Homo sapiens 58 cttcaacagt gcccaaggtgctcaa 25 59 49 DNA Homo sapiens modified_base (1)..(1) Biotinylated 59ttagggaacc ctcactgaat gaatgaatga atgaatgaat gaatgaatg 49 60 25 DNA Homosapiens 60 cattcattca ttcagtgagg gttcc 25 61 48 DNA Homo sapiensmodified_base (1)..(1) Biotinylated 61 taatacgact cactataggg agacatcgacaaggtgcgct tcctggag 48 62 22 DNA Homo sapiens 62 cgcgggcttt gtcgttggttag 22 63 20 DNA Homo sapiens modified_base (1)..(1) Biotinylated 63caggatctta ttctgctgct 20 64 48 DNA Homo sapiens 64 taatacgact cactatagggagaccccttt ttgtccccca actggaga 48 65 20 DNA Homo sapiens modified_base(1)..(1) Biotinylated 65 ccaaaagcct tcatacatct 20 66 32 DNA Homo sapiens66 aaggtgtgca cttttattca actggtctca ag 32 67 49 DNA Homo sapiens 67taatacgact cactataggc tggctggttt attgctgata aatctggag 49 68 60 DNA Homosapiens 68 tttttttttt tttttttttt tttttttttt ccaatgctta atcagtgaggcacctatctc 60 69 21 DNA Homo sapiens modified_base (1)..(1) Biotinylated69 cgagacccac gctcaccggc t 21 70 48 DNA Homo sapiens 70 taatacgactcactataggg cacccagaaa cgctggtgaa agtaaaag 48 71 51 DNA Homo sapiensmodified_base (1)..(1) Biotinylated 71 taatacgact cactataggg agaggaaaactgggtgcaga gggttctgga g 51 72 24 DNA Homo sapiens 72 ggcaaccctacaacagaccc acac 24 73 20 DNA Homo sapiens modified_base (1)..(1)Biotinylated 73 agccctcttc aaaaacttct 20

We claim:
 1. A method of providing an internal control for an individual test site in a nucleic acid hybridization reaction assay to determine the presence of at least one nucleic acid sequence of interest in at least one nucleic acid containing sample, wherein the nucleic acid hybridization assay is performed on an electronically controlled microarray comprising at least two test sites, the method comprising: (a) attaching a nucleic acid probe mix consisting of a first nucleic acid probe specific for a first internal control nucleic acid sequence known to be present in the sample, and a second nucleic acid probe specific for a second nucleic acid sequence of interest to a first test site on the electronically controlled microarray; (b) attaching a mixed nucleic acid probe consisting of the first nucleic acid probe and a third nucleic acid probe specific for a third nucleic acid sequence of interest, wherein the third nucleic acid sequence of interest may be the same as or different than the second nucleic acid sequence of interest, to a second test site on the electronically controlled microarray; (c) electronically hybridizing the sample nucleic acids from at least one sample to the nucleic acid probes on the first and second test sites; (d) specifically detecting the extent of hybridization of the sample nucleic acids to the first nucleic acid probe at the first and second test sites; (e) specifically detecting the extent of hybridization of the sample nucleic acids to the second and third nucleic acid probes at the first and second test sites; (f) comparing the hybridization values obtained for the first nucleic acid probe at the first and second test sites to obtain a normalization factor; and (g) normalizing the hybridization values obtained in (e) for the second and third probes using the normalization factor obtained in (f).
 2. The method of claim 1 wherein the first nucleic acid sequence is a sequence which encodes, or is complementary to a sequence which encodes, a housekeeping gene.
 3. The method of claim 1 wherein the first nucleic acid sequence is an exogenous nucleic acid sequence which has been added to the sample.
 4. The method of claim 1 wherein the specific detection in (d) is by detecting a labeled nucleotide which has been specifically incorporated by a nucleic acid polymerase reaction into the sample nucleic acids which contain the first nucleic acid sequence.
 5. The method of claim 1, further comprising the step of electronically or passively hybridizing a first reporter nucleic acid comprising a detectable moiety specific for the first nucleic acid sequence to the sample nucleic acids which are hybridized to the first nucleic acid probe at the first and second test sites, wherein the specific detection in (d) is by detecting the detectable moiety.
 6. The method of claim 1, further comprising the step of extending the first nucleic acid probe by utilizing the sample nucleic acids which have hybridized to the first nucleic acid probe as a template for a nucleic acid polymerase reaction, wherein the specific detection in (d) is by detecting a labeled nucleotide which has been incorporated into the extended first nucleic acid probe by the polymerase reaction.
 7. The method of claim 6, wherein the second and third nucleic acid probes attached in (a) and (b) comprise a protecting group that prevents enzymatic extension of the probes.
 8. The method of claim 1 wherein the specific detection in (e) is by detecting a labeled nucleotide which has been specifically incorporated by a nucleic acid polymerase reaction into the sample nucleic acids which contain the second and third nucleic acid sequences of interest.
 9. The method of claim 1, further comprising the step of electronically or passively hybridizing one or more reporter nucleic acid probes comprising a detectable moiety to the sample nucleic acids which are hybridized to the second and third nucleic acid probes at the first and second test sites, wherein the reporter probes are specific for the second and third nucleic acid sequences of interest and wherein the specific detection in (e) is by detecting the detectable moiety.
 10. The method of claim 1, further comprising the step of extending the second and third nucleic acid probes by utilizing the sample nucleic acids which have hybridized to the second nucleic acid probe as a template for a nucleic acid polymerase reaction, wherein the specific detection in (e) is by detecting a labeled nucleotide which has been incorporated into the extended second and third nucleic acid probes by the polymerase reaction.
 11. The method of claim 10, wherein the first nucleic acid probes attached in (a) and (b) comprise a protecting group that prevents enzymatic extension of the probes.
 12. The method of claim 1, wherein a first detectable moiety is detected in step (d), and a second detectable moiety is detected in step (e).
 13. The method of claim 12 wherein the first and second detectable moieties are independently selected from the group consisting of fluorescent moieties, colorigenic moieties, chemiluminescent moieties, and affinity moieties.
 14. The method of claim 13 wherein the first and second detectable moieties are fluorescent moieties. 